There is a continuous need in medical practice, research and diagnostic procedures for rapid, accurate and qualitative or quantitative determinations of biological substances which are present in biological fluids at low concentrations. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, prostaglandins or infectious organisms in blood, urine, saliva, vaginal secretions, dental plaque, gingival crevicular fluid and other biological specimens has to be determined in an accurate and rapid fashion for suitable diagnosis or treatment.
To provide such determinations, various methods have been devised for isolating and identifying biological substances employing specific binding reactions between the substance to be detected (identified herein as a "specific binding ligand") and a compound specifically reactive with that substance (sometimes identified as a "receptor" for the ligand).
The complex formed between ligand and receptor can be detected by a variety of known methods. The most commonly used methods employ a signal generating moiety of some type which is either already attached to one of the components of the complex, or becomes part of the complex through further reaction. For example, in the formation of a complex of biotin with avidin, the complex may be detected using a label on either the avidin or biotin molecule. Such a label can be a radioisotope or an enzyme conjugated with the avidin or biotin. Alternatively, the avidinbiotin complex might be detected by further reaction with a labeled molecule which is specific to either or both parts of the complex. It is commonly known to do the same with antigens and their corresponding antibodies.
Preferred labels in specific binding reactions are clearly enzymes because the handling and disposal problems associated with radioisotopes can be avoided. Many enzymes are known to be useful in this context with peroxidases being the most common.
In diagnostic tests designed to be rapid and easy to use with moderate training in a doctor's office or clinic, the specific binding ligand of interest (such as an antigen from an infectious agent) is often detected using colorimetric, fluorescent or chemiluminescent signals resulting from reaction of the enzyme label with its corresponding substrate. There is a need to produce the signal quickly and intensely if the ligand is present.
However, there is also a need to have the signal produced in a defined region of a test zone in a test device so an adjacent or surrounding region could be used as a background control. In such cases, production of the signal should be modulated or stopped after a certain period of time in order to provide clear distinction between test zone and background zone.
It is well known that sodium azide can be used to stop the production of detectable signal when peroxidase is used as a label in specific binding reactions. However, the use of sodium azide has problems associated with it, namely incomplete signal inhibition, toxicity and its explosive nature.
An advance in the art is provided by the invention described and claimed in copending U.S.S.N. 773,063 (filed on even date herewith by Contestable, Boyer, Snyder and Kissel) and entitled "Diagnostic Test Kit and Specific Binding Assay Using Modulator of Signal Resulting from Peroxidase Label". Our colleagues found that benzohydroxamic acid and similar compounds can effectively stop the formation of detectable signal if the acid is contacted with the specific binding complex after signal-producing reagents have been added. This advantageously avoided the use of sodium azide for this purpose.
We found, however, that in the development of certain very rapid specific binding assays, such as a sandwich assay for Streptococcus A, certain additional improvements were needed. In particular, we found that the time needed to add the acid as a "signal stop" reagent at the end made the assay undesirably long so that customer satisfaction was diminished. Moreover, we observed that the assay continued to show "false positive" signals from continued signal production if a "signal stop" reagent is not added.
Further still, in the highly competitive field of immunoassays designed for doctors' offices, there is a need to reduce the number of assay steps and reagents as much as possible to reduce operator error, assay cost and waste. It was considered important to eliminate the use of a "signal stop" reagent at the end of the assay if possible.